Project Summary/Abstract There is a growing need for veterinary and public health laboratories to perform whole genome sequencing for monitoring antimicrobial resistance and protecting the safety of people and animals. The FDA Vet-LIRN is currently assessing a small benchtop sequencing platform (Illumina iSeq) that could potentially be deployed across their network. Thus far, it has only been tested with Salmonella, and further methods optimization and evaluation is needed to ensure that the platform can be used by other laboratories that have less familiarity with sequencing technologies. E. coli is the most common pathogen tested for antibiotic susceptibility in veterinary diagnostic labs. It is also one of the most concerning for multi-drug resistance; several isolates captured thus far in Vet-LIRN active surveillance are predicted to be pan-resistant to all drugs used in human or animal medicine. Because the methods for susceptibility testing are not standardized, and specific drug panels are used for animal isolates that do not cover all antibiotics used in humans, it was not until these strains were sequenced that the severity was fully appreciated. Having sequencing capacity for this organism in more labs will facilitate rapid surveillance and better equip labs to participate in pet food contamination cases, which are now heavily focused on sequencing results. This directly supports the mission of FDA to ensure the safety of our nation's food supply and protect public health. Since E. coli is commonly found in people and animals, another critical need is the ability to discern the potential virulence of different strains using molecular subtyping. Unfortunately, no bioinformatic tools currently exist that will provide interpretations that are host specific for animals while still conforming to the pathogenic profiles used for humans. The capacity to perform advanced molecular characterization of bacterial isolates in a reliable manner is critical to our ability to respond quickly to suspected microbiological contamination and monitor antimicrobial resistance in animals and feed. The proposed studies will have important impacts for both human and veterinary health and will improve the capabilities of the Vet-LIRN to perform effective surveillance for food-borne pathogens.